METTL3介导Cry2 m6A甲基化修饰促进人肝细胞癌Huh-7细胞乐伐替尼耐药OA
METTL3-mediated Cry2 m6A methylation modification promotes lenvatinib resistance in human hepatocellular carcinoma Huh-7 cells
目的 探讨隐花色素蛋白2(Cry2)及其N6-甲基腺苷(m6A)甲基化修饰在人肝细胞癌(HCC)细胞乐伐替尼耐药中的作用.方法 体外培养人HCC细胞系Huh-7及其乐伐替尼耐药细胞株(Huh-7/LR).向Huh-7/LR细胞中转染si-Cry2、oe-Cry2、si-甲基转移酶样蛋白3(METTL3)、oe-METTL3以及干扰阴性对照(si-NC)和过表达空载(Vector)质粒,将未转染质粒的Huh-7/LR细胞作为对照.采用MTT法检测细胞对乐伐替尼的药物敏感性;流式细胞术检测各组细胞凋亡率;比色法检测细胞中RNA m6A甲基化水平;甲基化RNA免疫共沉淀法检测各组细胞中Cry2 RNA m6A甲基化水平;实时定量PCR和Western blotting检测各组细胞中Cry2和METTL3 mRNA和蛋白表达水平.结果 与Huh-7比较,Huh-7/LR细胞对乐伐替尼的敏感性显著降低(P<0.05),耐药指数为6.320;同时,Huh-7/LR细胞中Cry2 mRNA和蛋白表达水平较Huh-7细胞显著降低(P<0.01),m6A甲基化水平和METTL3蛋白表达水平显著升高(均P<0.05).过表达Cry2可显著增强Huh-7/LR细胞对乐伐替尼的敏感性,并促进细胞凋亡(P均<0.05).沉默METTL3可显著降低Huh-7/LR细胞Cry2 m6A甲基化水平(P<0.01),上调Cry2 mRNA和蛋白表达水平,提高Huh-7/LR细胞对乐伐替尼的敏感性,促进细胞凋亡(P均<0.05),METTL3过表达则呈现相反的作用结果.然而,沉默Cry2可显著逆转METTL3基因沉默对Huh-7/LR细胞乐伐替尼的增敏作用(P<0.05).结论 METTL3介导Cry2 m6A甲基化水平升高导致Cry2表达下调,进而促进人HCC细胞乐伐替尼耐药.
Objective To investigate the effects of cryptochrome 2(Cry2)and its N6-methyladenosine(m6A)methylation modification on lenvatinib resistance in human hepatocellular carcinoma(HCC)cells.Methods Human HCC cell line Huh-7 and its lenvatinib-re-sistant cells(Huh-7/LR)were cultured in vitro.si-Cry2,oe-Cry2,si-methyltransferase-like protein 3(METTL3),oe-METTL3 interference negative control(si-NC),and empty overexpression vector(Vector)plasmids were transfected into Huh-7/LR cells.The control group con-sisted of Huh-7/LR cells that were not transfected with the plasmid.The drug sensitivity of each group was assessed using the MTT assay.The rate of apoptosis in each group was determined by flow cytometry.The methylation level of RNA m6A in the cells was detected using colorimetry.The m6A methylation levels of Cry2 in each group were analyzed using the methylation RNA immunoprecipitation method.The mRNA and protein expression levels of Cry2 and METTL3 in each group were measured by real-time quantitative PCR and Western blot-ting,respectively.Results When compared to Huh-7 cells,the sensitivity of Huh-7/LR cells to lenvatinib was found to be significantly decreased(P<0.05),resulting in a drug resistance index of 6.320.Simultaneously,there was a significant decrease seen in the mRNA and protein expression levels of Cry2 in these cells(P<0.01).Additionally,the methylation level of RNA m6A and protein expression level of METTL3 were significantly elevated(P<0.05).Overexpression of Cry2 significantly augmented the sensitivity of the Huh-7/LR cells to lenvatinib and facilitated apoptosis(all P<0.05).Silencing METTL3 notably attenuated the methylation of Cry2 m6A in Huh-7/LR cells(P<0.01),upregulated the mRNA and protein expression levels of Cry2,increased the sensitivity of Huh-7/LR cells to lenvatinib,and promoted apoptosis(all P<0.05).Conversely,overexpressing METTL3 exerted an opposing effect.However,silencing Cry2 effectively reversed the sensitizing effect of METTL3 silencing to lenvatinib in Huh-7/LR cells.Conclusion METTL3 facilitates the elevation of Cry2 m6A methylation levels,thus resulting in the downregulation of Cry2 expression and consequently promoting resistance to lenvatinib in human HCC cells.
张喜华;何前进;郝泉水;李珊;李秀芳
黄冈市中心医院 肝胆外科,湖北 黄冈 438000黄冈市中心医院 肝胆外科,湖北 黄冈 438000黄冈市中心医院 麻醉科,湖北 黄冈 438000黄冈市中心医院 麻醉科,湖北 黄冈 438000黄冈市中心医院 麻醉科,湖北 黄冈 438000
医药卫生
肝细胞癌乐伐替尼隐花色素蛋白2N6-甲基腺苷甲基化耐药
hepatocellular carcinomalenvatinibcryptochrome 2N6-methyladenosinedrug resistance
《中国医科大学学报》 2026 (5)
416-422,7
湖北省卫生健康委科研项目(WJ2021M086)
评论